Investigation into the abnormal regulation of muscle glycogen synthesis in insulin resistant subjects has revealed an association of amino acid polymorphisms at codons 883 and 905 in the G subunit of Protein Phosphatase 1 (PP1) with NIDDM. Translationally optimized expression plasmid constructs encoding the wild-type and the mutant protein were transiently transfected into mouse C2C12 muscle cell line. We observed an increased expression level of a protein with the expected size as compared to the vector transfected control cells. This protein coprecipitated with glycogen, as expected of the G subunit. The protocol for assaying glycogen synthase activity on the transfected C2C12 cells has been optimized. We are currently selecting stably transfected C2C12 clones that are suitable for characterizing the potentially different functions of the G subunit variants. The discovery of Protein Targeting to Glycogen as a novel PP1 binding protein that potentially regulates glycogen metabolism has led us to investigate its coding region by automated sequencing. No polymorphism is detected in 10 non-related extremely insulin resistant subjects compared to 1 insulin sensitive and 1 Caucasian controls. In a collaborative effort to enhance our understanding on insulin resistance, we are in the process of comparing gene expression of extreme insulin resistant and insulin sensitive subjects using genetic array technology. As a genome scan for linkage to obesity and energy metabolism has shown a high LOD score linkage of chromosome location 20q11.2 with 24 hour respiratory quotient (RQ), we investigated the coding region of Agouti Signalling Protein as a candidate gene which lies in the region. Comparative sequencing of 15 non-related subjects with lowest RQ and another 15 with highest RQ values revealed no polymorphism. Association analysis of markers in the region is underway. We are also investigating the potential relationship of obesity and a recently discovered protein located on chromosome 16q22, Agouti Related Protein.